When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z‐DNA have been diffused into this system and used to measure the amount of Z‐DNA present in the nuclei. It has previously been shown that the amount of Z‐DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z‐DNA in the c‐myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c‐myc gene are shown to form Z‐DNA when the cell is transcribing c‐myc. The Z‐DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c‐myc gene is down‐regulated, the Z‐DNA content goes to undetectable levels within 30–60 min.

Real-time quantitative PCR is used routinely for the high-throughput diagnosis of viral pathogens, such as West Nile virus (WNV). Rapidly evolving RNA viruses present a challenge for diagnosis because they accumulate mutations that may render them undetectable. To explore the effect of sequence variations on assay performance, we generated every possible single point mutation within the target region of the widely used TaqMan assay for WNV and found that the TaqMan assay failed to detect 47% of possible single nucleotide variations in the probe-binding site and was unable to detect any targets with more than two mutations. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green. The SYBR green-based assay was as sensitive as the TaqMan assay for WNV. Importantly, it detected 100% of possible WNV target region variants. The assay developed here adds an additional layer of protection to guard against false-negative results that result from natural variations or drug-directed selection and provides a rapid means to identify such variants for subsequent detailed analysis.

Kaposi’s sarcoma (KS), caused by KS-associated herpesvirus (KSHV), is the most common cancer among HIV-infected patients in Malawi and in the United States today. In Malawi, KSHV is endemic. We conducted a cross-sectional study of patients with HIV infection and KS with no history of chemo- or antiretroviral therapy (ART). Seventy patients were enrolled. Eighty-one percent had T1 (advanced) KS. Median CD4 and HIV RNA levels were 181 cells/mm3 and 138,641 copies/ml, respectively. We had complete information and suitable plasma and biopsy samples for 66 patients. For 59/66 (89%) patients, a detectable KSHV load was found in plasma (median, 2,291 copies/ml; interquartile range [IQR], 741 to 5,623). We utilized a novel KSHV real-time quantitative PCR (qPCR) array with multiple primers per open reading frame to examine KSHV transcription. Seventeen samples exhibited only minimal levels of KSHV mRNAs, presumably due to the limited number of infected cells. For all other biopsy samples, the viral latency locus (LANA, vCyc, vFLIP, kaposin, and microRNAs [miRNAs]) was transcribed abundantly, as was K15 mRNA. We could identify two subtypes of treatment-naive KS: lesions that transcribed viral RNAs across the length of the viral genome and lesions that displayed only limited transcription restricted to the latency locus. This finding demonstrates for the first time the existence of multiple subtypes of KS lesions in HIV- and KS-treatment naive patients.

MicroRNAs are regulated by gene alteration, transcription, and processing. Thus far, few studies have simultaneously assessed all 3 levels of regulation. Using real-time quantitative polymerase chain reaction (QPCR)-based arrays, we determined changes in gene copy number, pre-miRNA, and mature miRNA levels for the largest set of primary effusion lymphomas (PELs) to date. We detected PELspecific miRNA gene amplifications, and concordant changes in pre-miRNA and mature miRNA. We identified 68 PELspecific miRNAs. This defines the miRNA signature of PEL and shows that transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant information that can be used for the classification of human tumors. Most miRNA gene loci are interspersed between coding regions or located within introns, though some can be embedded within an open reading frame. Thirty-seven percent of human miRNAs are organized in multi-miRNA clusters, 3 many of which can be found around fragile sites. 4,5 Clustered miRNAs are regulated by a common promoter and processed from a single primary transcript (pri-miRNA) that may contain several miRNAs, as well as coding exons. Hence, miRNAs are subject to (1) genomic alterations at the DNA level, (2) transcriptional regulation at the pre-miRNA level, and (3) processing control at the mature miRNA level. Thus far, few studies have evaluated these 3 modes of regulation simultaneously.In the nucleus, Drosha initiates miRNA processing ( Figure 1A) by cleaving the primary miRNA (pri-miRNA) to release the approximately 60-nt-long precursor miRNAs (pre-miRNAs). 6 After export to the cytoplasm, the pre-miRNAs are further processed by Dicer to yield an approximately 22-bp miRNA duplex. 7,8 One strand of this duplex is then incorporated into the RNA-induced silencing complex (RISC), where it guides the RISC to mRNAs bearing complementary sequences. 9,10 If the mRNA contains a perfectly complementary sequence, the RISC component Ago2 cleaves the target leading to mRNA degradation. 11,12 In the case of an imperfect complementary target, RISC binding can induce translational inhibition. 12 Translation inhibition is highly cooperative and requires several RISCs, potentially each with a different miRNA. 13,14 We hypothesized that cancer-specific miRNA profiles are determined by a combination of changes at the level of the gene locus, that is, mutations, deletions, or amplifications, changes at the level of transcriptional regulation, and changes at the level of miRNA processing. Hence, comprehensive miRNA profiling should query genomic loci, precursor miRNAs, as well as mature miRNAs. We hypothesized further that this information can be used for the differential diagnosis of lymphomas, specifically of primary effusion lymphoma (PEL).PELs are a unique type of post-germinal center diffuse large B-cell lymphoma (DLBCL). 15,16 Clinically PELs are defined by their effusion phenotype. Furthermore, all PEL tumors carry Kaposi sarcoma-associated herpesvirus (KSHV). KSHV also encodes viral miRNAs. 1...

Quantitative real-time PCR has become the method of choice for measuring mRNA transcription. Recently, fast PCR protocols have been developed as a means to increase assay throughput. Yet it is unclear whether more rapid cycling conditions preserve the original assay performance characteristics. We compared 16 primer sets directed against Epstein–Barr virus (EBV) mRNAs using universal and fast PCR cycling conditions. These primers are of clinical relevance, since they can be used to monitor viral oncogene and drug-resistance gene expression in transplant patients and EBV-associated cancers. While none of the primers failed under fast PCR conditions, the fast PCR protocols performed worse than universal cycling conditions. Fast PCR was associated with a loss of sensitivity as well as higher variability, but not with a loss of specificity or with a higher false positive rate.